Role of aspartate-133 and histidine-458 in the mechanism of tryptophan indole-lyase from Proteus vulgaris

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.     

Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.     

A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis

A glutathione monomer has been docked into the active site cleft of T4 glutaredoxin (previously called T4 thioredoxin) using molecular graphics. The central part of the cleft is formed by the side chain of Tyr-16 on one side and the residues Thr-64, Met-65, and Pro-66 on the other. The entire glutathione molecule fits well into the cleft. A cis-peptide bond between the residues Met-65 and Pro-66 allows glutathione to bind in an anti-parallel fashion to residues 64-66. Hydrogen bonds can be formed between Met-65 and the glutathione cysteine. This binding positions the glutathione sulfur atom ideally for reaction with the glutaredoxin disulfide. In the model, glutathione can form a hydrogen bond to the hydroxyl group of Tyr-16. Charged interactions at opposite ends of the binding cleft are provided by His-12 and Asp-80. The negatively charged alpha-carboxyl group of glutathione may interact with a positive helix dipole of the protein. Fifteen mutant T4 glutaredoxins have been produced and assayed for glutathione binding by determining thioltransferase activity. Mutant proteins with substitutions in the sides of the cleft (Tyr-16, Pro-66) exhibited the most marked decreases in thioltransferase activity. Mutation of His-12 to a serine decreases the catalytic efficiency whereas substitution of Asp-80 by serine increases the catalytic efficiency. A double mutant, D80S;H12S, has much less affinity for glutathione than either single mutant. Substitution of Cys-14 produces an inactive protein, whereas C17S retains some thioltransferase activity.     

Mutational analysis of basic residues in the rat vesicular acetylcholine transporter. Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation

The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.     

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.     

A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm. A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.     

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.     

Location of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis

In addition to binding Ca(2+), the S100 protein S100B binds Zn(2+) with relatively high affinity as confirmed using isothermal titration calorimetry (ITC; K(d) = 94 +/- 17 nM). The Zn(2+)-binding site on Ca(2+)-bound S100B was examined further using NMR spectroscopy and site-directed mutagenesis. Specifically, ITC measurements of S100B mutants (helix 1, H15A and H25A; helix 4, C84A, H85A, and H90A) were found to bind Zn(2+) with lower affinity than wild-type S100B (from 2- to >25-fold). Thus, His-15, His-25, Cys-84, His-85, and perhaps His-90 of S100B are involved in coordinating Zn(2+), which was confirmed by NMR spectroscopy. Previous studies indicate that the binding of Zn(2+) enhances calcium and target protein-binding affinities, which may contribute to its biological function. Thus, chemical shift perturbations observed here for residues in both EF-hand domains of S100B during Zn(2+) titrations could be detecting structural changes in the Ca(2+)-binding domains of S100B that are pertinent to its increase in Ca(2+)-binding affinity in the presence of Zn(2+). Furthermore, Zn(2+) binding causes helix 4 to extend by one full turn when compared to Ca(2+)-bound S100B. This change in secondary structure likely contributes to the increased binding affinity that S100B has for target peptides (i.e., TRTK peptide) in the presence of Zn(2+).     

Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins

Heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2alpha, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory Cys-Pro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.     

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.     

The conserved His-144 in the PsbP protein is important for the interaction between the PsbP N-terminus and the Cyt b559 subunit of photosystem II

The PsbP protein regulates the binding properties of Ca(2+) and Cl(-), and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl(-) anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S(1)/S(2) transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b(559) α subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.     

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.     

Structural diversity within the mononuclear and binuclear active sites of N-acetyl-D-glucosamine-6-phosphate deacetylase

NagA catalyzes the hydrolysis of N-acetyl-d-glucosamine-6-phosphate to d-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-d-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the alpha-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.     

Characterization of the metalloactivation domain of an arsenite/antimonite resistance pump

The ArsAB extrusion pump encoded by the ars operon of Escherichia coli plasmid R773 confers resistance to the toxic trivalent metalloids arsenite [As(III)] and antimonite [Sb(III)]. The ArsA ATPase, the catalytic subunit of the pump, has two homologous halves, A1 and A2. At the interface of these two halves are two nucleotide-binding domains and a metalloid-binding domain. Cys-113 and Cys-422 have been shown to form a high-affinity metalloid binding site. The crystal structure of ArsA shows two other bound metalloid atoms, one liganded to Cys-172 and His-453, and the other liganded to His-148 and Ser-420. The contribution of those putative metalloid sites was examined. There was little effect of mutagenesis of residues His-148 and Ser-420 on metalloid binding. However, a C172A ArsA mutant and C172A/H453A double mutant exhibited significantly decreased affinity for Sb(III). These results suggest first that there is only a single high-affinity metalloid binding site in ArsA, and second that Cys-172 controls the affinity of this site for metalloid and hence the efficiency of metalloactivation of the ArsAB efflux pump.     

The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Mutational analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).     

Stability of TEM β-lactamase mutants hydrolyzing third generation cephalosporins

The stability properties of six natural mutants of the TEM-1 β-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the Nδ of His-164 and the Oδ of Asp-179. The properties of the G238S+E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon. © 1995 Wiley-Liss, Inc.     

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.